Chemical and biological samples often contain mixtures of compounds. A variety of chromatographic techniques for separation of mixtures have been developed, and many systems for chromatographic separation and purification are commercially available.
Among the best-known chromatographic techniques are gas chromatography, high performance liquid chromatography (HPLC) and super-critical fluid chromatography (SFC). HPLC methods can be used to separate polar and non-polar compounds; the solvent (or mobile phase) and stationary phase to be used in an HPLC method are chosen based upon the types of analytes to be separated. With careful selection of the mobile phase and stationary phase, many mixtures can be separated into well-resolved peaks or fractions which can be isolated for further analysis or use. Characterization by methods such as mass spectrometry (MS) provides information about the analytes present in the sample.
A variety of HPLC techniques have been reported. Among the most widely-used are “normal phase” HPLC (generally useful for relatively polar analytes; least-polar analytes usually elute first) and “reversed phase” HPLC (RP-HPLC, generally used for less polar analytes; least-polar analytes generally elute last). A variation known as “hydrophilic interaction chromatography” or HILIC is useful for highly polar analytes that would not be sufficiently retained on a reversed-phase column.
However, when complex mixtures are involved, a single chromatographic (e.g., HPLC) separation may not be capable of separating all of the compounds into well-separated peaks or fractions. If peaks are not well-resolved, impurities or contaminants may be present even after separation, interfering with characterization of a collected fraction. To address this problem, multi-dimension chromatographic methods have been developed. In these methods, a sample is subjected to a first separation. The solvent stream resulting from the first separation is typically collected in fractions representing partially-purified compound mixtures; individual fractions are then selected and subjected to a second separation technique. The conditions of the first and second separations are generally different, and, if chosen carefully, the second separation should permit the separation of compounds which were not resolved in the first separation dimension. Examples of such multi-dimensional chromatographic methods include, e.g., Tranchida, P Q et al. J. Chromatogr A. 1054(1-2):3-16 (2004).
Such methods often involve the use of different columns and different mobile phases in each of the two chromatographic methods, which can result in added complexity. For example strong-cation exchange (SCX) separation followed by RP-HPLC has been used to analyze peptide mixtures. However, the high salt concentrations and/or organic solvents often required by the SCX conditions may not be compatible with the conditions required for RP-HPLC, and additional sample work-up is often required.
Furthermore, the mobile phases or mobile phase additives used in the two separations may not be compatible with detectors, including mass spectrometers, or other parts of the analytical system, leading to additional difficulty in detection or sample processing.